This invention relates to the following features.
1. A cell line derived from human hepatic carcinoma capable of stably expressing human cytochromes P450.
2. (1) A method for analyzing an enzyme participating in the metabolism of a xenobiotic and/or an endogenous substrate, (2) a method for analyzing a metabolic pathway of a xenobiotic and/or an endogenous substrate, (3) a method for analyzing a chemical structure of the metabolite of a xenobiotic and/or an endogenous substrate, (4) a method for preparing the metabolite of a xenobiotic and/or an endogenous substrate, (5) a method for analyzing inhibition of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (6) a method for analyzing an accelerated activity of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (7) a method for analyzing expression of cytotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (8) a method for analyzing expression of genotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (9) a method for analyzing expression of carcinogenicity by the metabolism of a xenobiotic and/or endogenous substrate, (10) a method for analyzing mutagenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (11) a method for analyzing expression of hepatotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, and (12) a method for analyzing a xenobiotic and/or an endogenous substrate that acts on the liver, each method comprising use of the cell line according to (1).
3. (1) A method for screening a substance capable of inhibiting a xenobiotic and/or an endogenous substrate, (2) a method for screening a substance capable of accelerating the activity of a metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (3) a method for screening a substance capable of expressing cytotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (4) a method for screening a substance capable of expressing genotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (5) a method for screening a substance capable of expressing carcinogenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (6) a method for screening a substance capable of expressing mutagenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (7) a method for screening a substance capable of expressing hepatotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (8) a method for screening a xenobiotic and/or an endogenous substrate which acts on the liver, and (9) a method for screening a substance capable of acquiring a new physiological activity or increasing or decreasing the inherent physiological activity, through the metabolism of a xenobiotic and/or an endogenous substrate, each method comprising use of the cell line according to 1.
4. A compound or its salt obtainable using the screening method according to 3.
Hepatocytes are known to have a great many physiological functions, all of which play a very important function in terms of the metabolism of xenobiotics and/or endogenous substrates such as drugs, food additives, environmental pollutants, industrial chemicals and the like. At the same time, the function of metabolizing xenobiotics and/or endogenous substrates might lead to inducing the inhibition of metabolizing enzymes for xenobiotics and/or endogenous substrates by xenobiotics and/or endogenous substrates, to accelerate the activity of metabolizing enzymes for xenobiotics and/or endogenous substrates, to express cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, to express genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, to express carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, to express mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, to express hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, and so on. For these reasons, the function of xenobiotics and/or endogenous substrates has been widely studied. It is known that many enzymes are associated with the metabolism of xenobiotics and/or endogenous substrates referred to herein. Examples of such enzymes include UDP-glucuronosyltransferase, sulfotransferase, glutathione transferase, epoxy hydratase, N-acetyltransferase, flavin monooxygenase and cytochromes P450. Also, the presence of a cytochrome P450 reductase is crucial for expressing the enzymatic function of cytochromes P450. Of an array of these enzymes, cytochromes P450 play the most important role in the metabolism of xenobiotics and/or endogenous substrates. The term cytochromes P450 collectively refers to a class of enzymes including a great many molecular species. In the metabolism of xenobiotics and/or endogenous substrates in human liver, ten (10) species of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 are considered important. Also, these enzymes, which are distributed in human liver, are functionally different depending on species and hence, human-derived liver specimens are unable to be used as a stable test system. On the other hand, such a metabolic function of the liver involves a very strong specificity, i.e., differences in nature, depending on species, which makes it difficult to predict such diverse metabolic functions of human liver from experimental animals, e.g., rats. However, it is practically impossible to analyze these functions of interest in humans. For these reasons, human-derived cultured hepatocytes are considered useful not only in examining the function of human liver in a rapid, inexpensive, safe and accurate way provided in place of experimental animals, but also in producing a so-called artificial liver as a functional substitute for human liver. However, it is impossible to subculture normal human hepatocytes separated from tissues in vivo. Cells that can be established as a cell line often lack the differentiation capability possessed inherently and in most cases, do not exactly reflect the function of tissues to which the cell line originally belongs. A family of enzymes that metabolize xenobiotics and/or endogenous substrates especially in liver cells, among others, the family of cytochromes P450 molecular species loses its activity in an extremely short period of time in primary culture; any cell line that fully retains the property has not been found so far (J. Dich et al., Hepatology, 8, 39-45 (1988)). Thus, in light of the foregoing, there is an extensive need for hepatocytes that can retain the capability of metabolizing xenobiotics and/or endogenous substrates and can be incubated.
To date, however, no cultured cell line has been obtained as retaining the function associated with the metabolism of xenobiotics and/or endogenous substrates as in the liver. Particularly because the activity of cytochromes P450 is widely recognized to be rapidly lost in cultured cells, it has been hitherto attempted to stably express cytochromes P450 in the established cultured cells and by this, take over the metabolizing function of liver (M. Sawada et al., Mutation Research, 411, 19-43 (1998)). However, as stated above, the cell line for expression of cytochromes P450 should indispensably be derived from human liver cells. In addition, the activity of NADPH cytochromes P450 reductase is required for expressing the activity of cytochromes P450, requiring further expression of many more enzymes. Therefore, stable and safe reproduction of the metabolizing function in human liver should be in human-derived cultured hepatocytes that retain the activity of enzymes participating in the metabolism of cytochromes P450 as well as various other metabolisms.
As examples of the expression of cytochromes P450 in cells retaining the activity of various enzymes participating in metabolism, there are cases in which P450 was expressed in HepG2 cells using vaccinia virus (Methods in Enzymology, T. Aoyama et al. in Methods in Enzymology, 260, 85-92, edited by M. R. Waterman, Academic Press, 1991) and in which CYP2E1 was expressed in HepG2 cells (Y. Dai et al., Biochemistry, 32, 6928-6937, 1993). In the former case, careful handling is required, which is an obstacle to practical application. The latter was attempted for CYP2E1 alone but so far has not been attempted for many other species of cytochromes P450 present in the liver. Accordingly, if a cultured cell line that can retain the activity of a family of enzymes participating in the metabolism of xenobiotics and/or endogenous substrates in the liver could be obtained, this would enable a practitioner to (1) analyze an enzyme participating in the metabolism of xenobiotics and/or endogenous substrates, (2) analyze a metabolic pathway of xenobiotics and/or endogenous substrates, (3) analyze a chemical structure of the metabolite of xenobiotics and/or endogenous substrates, (4) prepare the metabolite of xenobiotics and/or endogenous substrates, (5) analyze inhibition of the metabolizing enzyme for xenobiotics and/or endogenous substrates, (6) analyze an accelerated activity of the metabolizing enzyme for xenobiotics and/or endogenous substrates, (7) analyze expression of cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (8) analyze expression of genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (9) analyze expression of carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, (10) analyze mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, (11) analyze expression of hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, and (12) analyze xenobiotics and/or endogenous substrates that act on the liver. Furthermore, this would enable a practitioner to (1) screen a substance capable of inhibiting xenobiotics and/or endogenous substrates, (2) screen a substance capable of accelerating the activity of metabolizing enzymes for xenobiotics and/or endogenous substrates, (3) screen a substance capable of expressing cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (4) screen a substance capable of expressing genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (5) screen a substance capable of expressing carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, (6) screen a substance capable of expressing mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, (7) screen a substance capable of expressing hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (8) screen xenobiotics and/or endogenous substrates which act on the liver, and (9) screen a substance capable of acquiring a new physiological activity or increasing or decreasing the inherent physiological activity, through the metabolism of xenobiotics and/or endogenous substrates. Thus, specific compounds or salts thereof, etc. can be obtained using the method for analysis and/or the method for screening above.
An object of this invention is to provide a cultured cell line derived from human liver, thereby to separate and produce the cell line that can stably express human cytochromes P450 CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4.
These cells enable a practitioner of the invention to (1) analyze an enzyme participating in the metabolism of xenobiotics and/or endogenous substrates, (2) analyze a metabolic pathway of xenobiotics and/or endogenous substrates, (3) analyze a chemical structure of the metabolite of xenobiotics and/or endogenous substrates, (4) prepare the metabolite of xenobiotics and/or endogenous substrates, (5) analyze inhibition of the metabolizing enzyme for xenobiotics and/or endogenous substrates, (6) analyze an accelerated activity of the metabolizing enzyme for xenobiotics and/or endogenous substrates, (7) analyze expression of cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (8) analyze expression of genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (9) analyze expression of carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, (10) analyze mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, (11) analyze the expression of hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, and (12) analyze xenobiotics and/or endogenous substrates that act on the liver. The cells further enable a practitioner of the invention to (1) screen a substance capable of inhibiting xenobiotics and/or endogenous substrates, (2) screen a substance capable of accelerating the activity of metabolizing enzymes for xenobiotics and/or endogenous substrates, (3) screen a substance capable of expressing cytotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (4) screen a substance capable of expressing genotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (5) screen a substance capable of expressing carcinogenicity by the metabolism of xenobiotics and/or endogenous substrates, (6) screen a substance capable of expressing mutagenicity by the metabolism of xenobiotics and/or endogenous substrates, (7) screen a substance capable of expressing hepatotoxicity by the metabolism of xenobiotics and/or endogenous substrates, (8) screen xenobiotics and/or endogenous substrates which act on the liver, and (9) screen a substance capable of acquiring a new physiological activity or increasing or decreasing the inherent physiological activity, through the metabolism of xenobiotics and/or endogenous substrates. Thus, particular compounds or salts thereof, etc. can be obtained, using the method for analysis and/or the method for screening.
In view of the foregoing problems, the present inventors have made extensive studies. As a result, they have established stable transformants capable of stably expressing cytochromes P450 in a human hepatocarcinoma-derived (or hepatic carcinoma-derived) cell line with an enhanced activity for participation in the metabolism of xenobiotics and/or endogenous substrates. The following further studies have resulted in accomplishing this invention.
That is, the present invention relates to the following features.
(1) A cell line derived from human hepatic carcinoma capable of stably expressing human cytochromes P450.
(2) The cell line according to (1), wherein human cytochromes P450 are capable of stably expressing CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 or CYP3A4.
(3) The cultured cell line according to (1), wherein the human hepatic carcinoma cell is HepG2.
(4) The cell line according to (1), which is Hepc/1A1.4, Hepc/1A2.9, Hepc/2B6.68, Hepc/2C8.46, Hepc/2C9.1, Hepc/2C19.12, Hepc/2D6.39, Hepc/2E1.3-8 or Hepc/3A4.5.
(5) A method for analyzing (a) an enzyme participating in the metabolism of a xenobiotic and/or an endogenous substrate, (b) a metabolic pathway of a xenobiotic and/or an endogenous substrate, (c) a chemical structure of the metabolite of a xenobiotic and/or an endogenous substrate, (d) inhibition of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (e) an accelerated activity of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate. (f) cytotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate. (g) genotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (h) carcinogenicity by the metabolism of a xenobiotic and/or endogenous substrate, (i) mutagenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (j) hepatotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, or (k) a xenobiotic and/or an endogenous substrate that acts on the liver.
(6) A method for preparing the metabolite of a xenobiotic and/or an endogenous substrate, which comprises using the cell line according to (1).
(7) A method for screening a substance which comprises using the cell line according to (1), wherein the substance is (a) a substance capable of inhibiting a xenobiotic and/or an endogenous substrate, (b) a substance capable of accelerating an activity of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (c) a substance capable of expressing cytotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (d) a substance capable of expressing genotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (e) a substance capable of expressing carcinogenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (f) a substance capable of expressing mutagenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (g) a substance capable of expressing hepatotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (h) a xenobiotic and/or an endogenous substrate which acts on the liver, or (i) a substance capable of acquiring a new physiological activity or increasing or decreasing the inherent physiological activity, through the metabolism of a xenobiotic and/or an endogenous substrate.
(8) A compound or its salt which is obtainable using the method according to (7).
(9) A pharmaceutical composition comprising the compound or its salt according to (8).
(10) A method for analysis which comprises using at least two cultured cell lines derived from human liver capable of stably expressing at least one of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, said analysis being for (a) an enzyme that participates in the metabolism of a xenobiotic and/or an endogenous substrate, (b) a metabolic pathway of a xenobiotic and/or an endogenous substrate, (c) a chemical structure of the metabolite of a xenobiotic and/or an endogenous substrate, (d) inhibition of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (e) an accelerated activity of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (f) cytotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (g) genotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (h) carcinogenicity by the metabolism of a xenobiotic and/or endogenous substrate, (i) mutagenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (j) hepatotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, or (k) a xenobiotic and/or an endogenous substrate that acts on the liver.
(11) A method for preparation of the metabolite of a xenobiotic and/or an endogenous substrate, which comprises using at least two cultured cell lines from human liver capable of stably expressing at least one of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4.
(12) A method for screening a substance which comprises using at least two cultured cell lines from human liver capable of stably expressing at least one of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, said substance being (a) a substance capable of inhibiting the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (b) a substance capable of accelerating an activity of the metabolizing enzyme for a xenobiotic and/or an endogenous substrate, (c) a substance capable of expressing cytotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (d) a substance capable of expressing genotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (e) a substance capable of expressing carcinogenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (f) a substance capable of expressing mutagenicity by the metabolism of a xenobiotic and/or an endogenous substrate, (g) a substance capable of expressing hepatotoxicity by the metabolism of a xenobiotic and/or an endogenous substrate, (h) a xenobiotic and/or an endogenous substrate which acts on the liver, or (i) a substance capable of acquiring a new physiological activity or increasing or decreasing the inherent physiological activity, through the metabolism of a xenobiotic and/or an endogenous substrate.
(13) A compound or a salt thereof, which is obtainable using the method according to (12).
(14) A pharmaceutical compound comprising a compound or a salt thereof according to (13).